Abstract

RNA virus infection is recognized by the RIG-I family of receptors that activate the mitochondrial adaptor MAVS, leading to the clearance of viruses. Antiviral signalling activation requires strict modulation to avoid damage to the host from exacerbated inflammation. Insulin receptor tyrosine kinase substrate (IRTKS) participates in actin bundling and insulin signalling and its deficiency causes insulin resistance. However, whether IRTKS is involved in the regulation of innate immunity remains elusive. Here we show that IRTKS deficiency causes enhanced innate immune responses against RNA viruses. IRTKS-mediated suppression of antiviral responses depends on the RIG-I-MAVS signalling pathway. IRTKS recruits the E2 ligase Ubc9 to sumoylate PCBP2 in the nucleus, which causes its cytoplasmic translocation during viral infection. The sumoylated PCBP2 associates with MAVS to initiate its degradation, leading to downregulation of antiviral responses. Thus, IRTKS functions as a negative modulator of excessive inflammation.

(a) IRTKS knockout mice display normal antibacterial activity. BMDMs and MEFs were subjected to immunoblotting with the indicated antibodies (left panel). IRTKS+/+ and IRTKS−/− mice were intraperitoneally administrated with Listeria (2 × 106) and survived mice were calculated in the following 7 days (right panel). n=20 mice per group. (b) IRTKS+/+ and IRTKS−/− mice were intranasally inoculated with VSV (5 × 105 p.f.u. for each mouse). Survival curves were calculated over infection. n=20. (c,d) IRTKS+/+ and IRTKS−/− mice were intranasally inoculated with VSV (5 × 105 p.f.u. for each mouse), followed by detection of serum interferons via an ELISA analysis. n=15. (e) Blood from mice treated as in b were collected and subjected to RNA extraction, followed by RT–PCR analysis with virus-specific primers. Virus values were normalized to that of β-actin. n=15. (f–h) IRTKS+/+ and IRTKS−/− mice were intranasally inoculated with VSV (5 × 105 p.f.u. for each mouse). IFN expression levels of peritoneal macrophages were examined at the indicated times (f and g). VSV mRNA from peritoneal macrophages was analysed through RT–PCR (h). For f–h, n=11. Data are shown as means±s.d. For (c–e), a two-way analysis of variance post hoc Bonferroni test was used; for f–h, a two-tailed unpaired Student's t-test was used. *P<0.05; **P<0.01; ***P<0.001. Data are representative of at least three independent experiments. p.f.u., plaque-forming unit.

(a) IRTKS+/+ and IRTKS−/− BMDMs were infected with the indicated viruses (m.o.i.=5) for 18 h. IFN levels were assayed through ELISA (upper panel) and IRF3 dimerization was examined by immunoblotting (lower panel). (b) IRTKS+/+ and IRTKS−/− BMDMs were infected with VSV (m.o.i.=5) for the indicated times, followed by RNA extraction and RT–PCR analysis of Ifnb. (c) IRTKS+/+ and IRTKS−/− BMDMs were infected with VSV-GFP (m.o.i.=5) for 24 h, followed by examination with confocal microscopy (upper panel). Cells were counterstained with DAPI for nucleus. GFP-positive cells were calculated (lower panel). Scale bar, 200 μm. (d) IRTKS+/+ and IRTKS−/− BMDMs were transfected with the indicated types of DNA or RNA (1 mg ml−1) for 18 h and analysed as a. (e) WT MEFs were transfected with IFN-β luciferase plasmid, various amounts of IRTKS and the indicated cDNAs involved in RNA sensing or GFP control for 24 h, followed by the analysis of promoter activity. (f) WT BMDMs were transfected with various amounts of IRTKS and the indicated cDNAs involved in RNA sensing or GFP control for 24 h, followed by RNA extraction for Ifnb expression (normalized to β-actin). (g) WT BMDMs expressing empty vector or IRTKS were incubated with VSV (m.o.i.=5) for the indicated times, followed by analysis of Ifnb mRNA (normalized to β-actin). (h,i) IRTKS−/− BMDMs with RIG-I or MAVS knockdown (h) were incubated with VSV (m.o.i.=5) for 12 h, followed by IFN examination through RT–PCR (i). Data are shown as means±s.d. For a,c,d–f,i), a two-tailed unpaired Student's t-test was used; for b,g, a two-way analysis of variance post hoc Bonferroni test was used. *P<0.05; **P<0.01; ***P<0.001. Data represent at least three separate experiments. m.o.i., multiplicity of infection.

(a) IRTKS+/+ and IRTKS−/− BMDMs were incubated with VSV (m.o.i.=5) for the indicated times, followed by immunoblotting with the indicated antibodies (upper panel). Ratios of MAVS/β-actin were calculated (lower panel). (b) IRTKS+/+ and IRTKS−/− BMDMs were incubated with VSV (m.o.i.=5) for the indicated times in the presence of 20 μg ml−1 CHX and 10 μM MG132, followed by immunoblotting with the indicated antibodies. (c) IRTKS+/+ and IRTKS−/− BMDMs were incubated with VSV (m.o.i.=5) for 16 h, followed by immunoprecipitation (IP) with anti-MAVS antibody. (d) Increasing amounts of Flag-tagged IRTKS and GFP control vector were co-transfected into BMDMs for 24 h. Protein levels were examined by immunoblotting with the indicated antibodies. (e) IRTKS promotes the degradation of MAVS through PCBP2. Flag-tagged IRTKS was transfected into scramble (shCtrl) or PCBP2-silenced BMDMs for 24 h, followed by immunoblotting with the indicated antibodies. (f) Flag-tagged IRTKS was co-transfected with WT MAVS or K2R-MAVS (K371R/K420R mutant) into shCtrl or PCBP2-silenced BMDMs for 24 h, followed by immunoblotting with the indicated antibodies. (g) PCBP2-depleted BMDMs were rescued with WT-PCBP2 or Δ1–81-PCBP2, followed by incubation with VSV (m.o.i.=5) for 16 h. Cell lysates were immunoblotted with the indicated antibodies. (h) WT MEFs were transfected with IFN-β luciferase plasmid, various amounts of PCBP2 and MAVS for 24 h, followed by analysis of promoter activity. (i) IRTKS+/+ and IRTKS−/− BMDMs were rescued with WT-IRTKS or Δ400–514-IRTKS, followed by incubation with VSV (m.o.i.=5) for 16 h. (j) WT MEFs were transfected with IFN-β luciferase plasmid, various amounts of IRTKS and MAVS for 24 h. Data are shown as means±s.d. For a, a two-way analysis of variance (ANOVA) post hoc Bonferroni test was used; for h,j, a one-way ANOVA followed by a Dunnett post hoc test was used. *P<0.05; **P<0.01; ***P<0.001. Data are representative of at least three independent experiments. m.o.i., multiplicity of infection.

IRTKS recruits Ubc9 that sumoylates PCBP2 in the nucleus right after VSV infection.

(a) Yeast strain AH109 was co-transfected with Gal4 DNA-binding domain (BD)-fused IRTKS and Gal4 activating domain (AD)-fused Ubc9. p53 and large T antigen were introduced as a positive control. (b) Mapping the interacting regions between IRTKS and Ubc9. The indicated GST-tagged IRTKS truncations were incubated with His-tagged Ubc9, followed by GST pull-down assay. (c) WT mice were intranasally inoculated with VSV (5 × 105 p.f.u. for each mouse) for the indicated times. Peritoneal macrophages were collected and lysed for immunoprecipitation (IP) with anti-IRTKS antibody. Immunoprecipitates were immunoblotted with the indicated antibodies. (d) WT BMDMs were incubated with VSV (m.o.i.=5) for 8 h with or without ginkgolic acid (GA, 20 μm), followed by IP with anti-PCBP2 antibody. Immunoprecipitates were detected with anti-SUMO2 antibody. (e) WT BMDMs were incubated with VSV (m.o.i.=5) for the indicated times with or without 40 nM leptomycin B, followed by cytoplasmic and nuclear separation. Lysates were immunoprecipitated with anti-PCBP2 antibody, followed by immunoblotting with the indicated antibodies. (f) WT BMDMs were incubated with VSV (m.o.i.=5) for 1 h, followed by immunostaining with the indicated antibodies. Nuclei were counterstained with DAPI. Scale bar, 10 μm. (g) Recombinant PCBP2 was incubated with the indicated recombinant proteins in the presence of 5 mM ATP, followed by IP with antibody against PCBP2. The immunoprecipitates were immunoblotted with the indicated antibodies. (h) IRTKS+/+ and IRTKS−/− BMDMs were incubated with VSV (m.o.i.=5) for 8 h, followed by IP with anti-PCBP2 antibody. Immunoprecipitates were detected with anti-SUMO2 antibody. (i) shCtrl- or Ubc9-silenced BMDMs were incubated with VSV (m.o.i.=5) for 8 h, followed by IP with anti-PCBP2 antibody. Data are shown as means±s.d. Data were repeated at least three times with similar results. m.o.i., multiplicity of infection; p.f.u., plaque-forming unit.

(a) IRTKS+/+ and IRTKS−/− BMDMs were incubated with VSV (m.o.i.=5) for 8 h with or without ginkgolic acid (GA) (20 μm), followed by immunostaining with the indicated antibodies. Percentages of cytoplasmic PCBP2 (cytoplasmic PCBP2/total PCBP2) were calculated (right panel). At least 200 cells were counted. Scale bar, 10 μm. (b) WT BMDMs were incubated with VSV (m.o.i.=5) for 8 h, followed by immunoprecipitation (IP) with anti-PCBP2 antibody using cytoplasmic or nuclear fractions. (c) WT BMDMs were incubated with VSV (m.o.i.=5) for 8 h with or without GA (20 μm), followed by cytoplasmic and nuclear separation. IP was performed using anti-PCBP2 antibody. Immunoprecipitates were detected with the indicated antibodies. (d) PCBP2+/+ and PCBP2−/− BMDMs rescued with WT or K37R-PCBP2 were incubated with VSV (m.o.i.=5) for 8 h, followed by immunostaining with the indicated antibodies. Percentages of cytoplasmic PCBP2 (cytoplasmic PCBP2/total PCBP2) were calculated (right panel). At least 200 cells were counted. Scale bar, 10 μm. (e) PCBP2+/+ and PCBP2−/− BMDMs rescued with the indicated PCBP2 plasmids were incubated with VSV (m.o.i.=5) for 8 h, followed by IP with anti-PCBP2. (f) PCBP2+/+ and PCBP2−/− MEFs were transfected with Flag-AIP4 and Myc-PCBP2 (K37R) for 24 h, followed by immunoblotting with the indicated antibodies. (g) IRTKS+/+ and IRTKS−/− MEFs were transfected with Flag-AIP4 for 24 h with or without 40 nM leptomycin B, followed by immunoblotting with the indicated antibodies. (h) shCtrl- or AIP4-silenced MEFs were transfected with Flag-IRTKS for 24 h, followed by immunoblotting with the indicated antibodies. Data are shown as means±s.d. Data are representative of at least three separate experiments. m.o.i., multiplicity of infection.

(a) PCBP2−/− BMDMs were transfected with WT- or K37R-PCBP2 for 18 h (upper panel), followed by infection with VSV (m.o.i.=5) for 18 h. IFN levels were detected by ELISA (lower panel). (b) PCBP2−/− BMDMs were transfected with WT- or K37R-PCBP2 for 18 h, followed by infection with VSV (m.o.i.=5) for the indicated times. VSV mRNA of BMDMs was analysed by RT–PCR. (c) IRTKS+/+ and IRTKS−/− BMDMs were incubated with VSV (m.o.i.=5) for 8 h, followed by immunostaining with the indicated antibodies. Percentages of cytoplasmic PCBP2 (cytoplasmic PCBP2/total PCBP2) were calculated (right panel). At least 200 cells were counted. Scale bar, 10 μm. (d) IRTKS+/+ and IRTKS−/− BMDMs rescued with WT or Δ1–230-IRTKS were incubated with VSV (m.o.i.=5) for 8 h, followed by immunostaining with the indicated antibodies. Percentages of cytoplasmic PCBP2 (cytoplasmic PCBP2/total PCBP2) were calculated (right panel). At least 200 cells were counted. Scale bar, 10 μm. (e) IRTKS+/+ and IRTKS−/− BMDMs were rescued with WT or Δ1–230-IRTKS, followed by infection with VSV-GFP (m.o.i.=5) for 24 h. Cells were counterstained with DAPI (left panel). GFP-positive cells were calculated (right panel). Scale bar, 200 μm. (f) IRTKS+/+ and IRTKS−/− BMDMs rescued with WT or Δ400–514-IRTKS were incubated with VSV (m.o.i.=5) for 8 h, followed by immunostaining with the indicated antibodies. Percentages of cytoplasmic PCBP2 (cytoplasmic PCBP2/total PCBP2) were calculated (right panel). At least 200 cells were counted. Scale bar, 10 μm. (g) IRTKS+/+ and IRTKS−/− BMDMs rescued with the indicated IRTKS plasmids were incubated with VSV (m.o.i.=5) for 8 h, followed by immunoprecipitation (IP) with anti-PCBP2 antibody using cytoplasmic or nuclear fractions. Immunoprecipitates or cell lysates were immunoblotted with the indicated antibodies. Data are shown as means±s.d. For a,e, a two-tailed unpaired Student's t-testwas used; for b, a one-way analysis of variance followed by a Dunnett post hoc test was used using PCBP2+/+ cells as controls.*P<0.05; **P<0.01; ***P<0.001. Data are representative of at least three separate experiments. m.o.i., multiplicity of infection.